Thesis

Isolation and characterization of a vasopressin-like molecule in the medicinal leech, Hirudo midcinalis

One of the major tasks of modem neurobiology is explaining at the neuron level how specific behaviors are produced. Recent work in our laboratory has identified a putative peptidergic neuron in the medicinal leech, Hirudo medicinalis, which we believe, uses an Arg-vasopressin-like peptide as its transmitter. However, before further characterization of this neuron can precede the vasopressin-like molecule needs to be isolated and its molecular structure identified. In the course of this work, we designed an acetone-based extraction of nerve cords, developed a dot-immunoblot assay (DIA) that can be used to monitor the molecule throughout the purification process, and used size exclusion chromatography (SEC) to partially purify the vasopressin-like molecule. Additionally, using enzyme-linked immunosorbent assays (ELISAs), preliminary results suggest that 1.47X10-5 pg/nerve cord is being extracted. Partially purified nerve cord extracts, whole nerve cords and crude leech CNS extracts, were also sent to Dr. Lingjun Li at the University of Wisconsin-Madison for matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-MS) analysis. Unfortunately, we were unable to sequence the vasopressin-like peptide from the partially purified SEC fractions due to a contamination problem. In addition, MALDI-MS sequencing from intact leech nerve cords or crude extracts were unsuccessful due to a large number of peaks.

One of the major tasks of modem neurobiology is explaining at the neuron level how specific behaviors are produced. Recent work in our laboratory has identified a putative peptidergic neuron in the medicinal leech, Hirudo medicinalis, which we believe, uses an Arg-vasopressin-like peptide as its transmitter. However, before further characterization of this neuron can precede the vasopressin-like molecule needs to be isolated and its molecular structure identified. In the course of this work, we designed an acetone-based extraction of nerve cords, developed a dot-immunoblot assay (DIA) that can be used to monitor the molecule throughout the purification process, and used size exclusion chromatography (SEC) to partially purify the vasopressin-like molecule. Additionally, using enzyme-linked immunosorbent assays (ELISAs), preliminary results suggest that 1.47X10-5 pg/nerve cord is being extracted. Partially purified nerve cord extracts, whole nerve cords and crude leech CNS extracts, were also sent to Dr. Lingjun Li at the University of Wisconsin-Madison for matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-MS) analysis. Unfortunately, we were unable to sequence the vasopressin-like peptide from the partially purified SEC fractions due to a contamination problem. In addition, MALDI-MS sequencing from intact leech nerve cords or crude extracts were unsuccessful due to a large number of peaks.

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